A Virus Detection Method for Biological Products Based on Indicator Cell Culture

Abstract

This invention relates to a method for detecting viruses in biological products based on indicator cell culture. The method includes the following steps: S1, freezing-thawing and centrifuging the sample of the biological product to be tested to prepare a sample processing solution; S2, matching the corresponding indicator cell line according to the source of the sample and the detection target, and culturing the indicator cells to a confluence of 30-60% in plates, then setting up NC, EC, PC, and IG groups, and inoculating them with the corresponding processing solutions for incubation; S3, after incubation, replacing the culture medium with complete medium and performing continuous culture for 21 or 28 days, with passage operations performed according to preset nodes during the culture period, and CPE observed throughout; S4, after the culture cycle, performing endpoint detection on each group of cells, including HAd, HA, immunofluorescence staining detection, and quantitative detection of reverse transcriptase activity; S5, performing method validation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for detecting viruses in biological products based on indicator cell culture. Background Technology

[0002] Biologics are core products in the modern biopharmaceutical field, encompassing categories such as vaccines, recombinant biological products, and cell bank preparations. Their production process requires key materials such as bovine serum, porcine-derived raw materials, and animal-derived cell matrices. Exogenous viral contamination is a core risk factor restricting the safety of biologics. Contaminating viruses can be introduced during the production process and transferred to the final product, posing serious clinical safety risks. Therefore, establishing compliant, sensitive, and comprehensive methods for detecting exogenous viruses is a core aspect of biologics quality control and safety evaluation. Currently, cell culture is the mainstream technology for detecting exogenous viruses in biologics, relying on indicator cell culture to observe cytopathic effects, hemoadsorption phenomena, or combining immunofluorescence techniques to determine viral contamination levels. However, existing virus detection technologies have several technical shortcomings: First, the indicator cell selection is limited, only able to detect specific viruses, and cannot simultaneously cover bovine, porcine, retroviruses, and broad-spectrum viral factors, resulting in limited detection coverage; second, detection indicators are used in isolation, often relying solely on single indicators such as cytopathic effects or immunofluorescence, which can easily lead to missed detections of low-titer viruses; third, the methodological validation system is incomplete, with most methods only conducting simple repeatability tests without simultaneously completing comprehensive validation of specificity, detection limit, intermediate precision, and robustness, making it difficult to meet the requirements of domestic and international regulations such as the Chinese Pharmacopoeia, ICH, and FDA; fourth, the standardization of sample pretreatment and cell passage processes is insufficient, resulting in low virus enrichment efficiency and further reducing detection sensitivity.

[0003] Chinese invention patent CN102043047B discloses an immunofluorescence virus detection method based on cell smears. This technology focuses solely on immunofluorescence as a single detection method, failing to combine it with multiple indicators such as cytopathic effects and hemoadsorption for joint assessment. Furthermore, it lacks a complete methodological validation system and cannot meet the high-throughput, high-sensitivity screening requirements for various types of exogenous viruses in biological products. In summary, existing virus detection technologies cannot meet the standardized detection needs of all categories and types of exogenous viruses in biological products. There is an urgent need to develop a standardized cell culture virus detection method with appropriate indicator cell composition, combined use of multiple detection indicators, and comprehensive methodological validation to improve the comprehensiveness, accuracy, and reliability of virus detection in biological products. Summary of the Invention

[0004] This invention provides a method for detecting viruses in biological products based on indicator cell culture, comprising the following steps: S1, Sample pretreatment: The biological product sample to be tested is subjected to freeze-thaw lysis and centrifugation to prepare the sample lysis supernatant; S2, Construction of indicator cells and seeding system: Based on the source of the sample and the detection target, the corresponding indicator cell line was matched. After the indicator cells were plated and cultured to a confluence of 30%~60%, the NC group (negative control group), EC group (experimental group), PC group (positive control group), and IG group (interference group) were set up. After seeding with the corresponding treatment solution, they were incubated. S3, Virus amplification culture: After incubation, replace with complete culture medium and culture continuously for 21 or 28 days. During the culture period, perform passage operations according to preset nodes and observe CPE (cytopathic effect) throughout the process. S4, Multiple endpoint detection: After the culture cycle is completed, endpoint detection is performed on each group of cells. The endpoint detection includes HAd (hemoadsorption assay), HA (hemagglutination assay), immunofluorescence staining detection, and quantitative detection of reverse transcriptase activity. S5, Result Judgment and Method Validation: Based on the multi-endpoint detection results, the positive and negative results of the samples are determined, and the validity of the detection method is confirmed through the preset methodological validation parameters.

[0005] Existing methods for detecting viruses in biological products suffer from problems such as crude sample pretreatment, lack of specificity in indicator cell matching, non-standard culture and passage procedures, and limited endpoint detection methods with incomplete methodological validation. These issues can easily lead to missed detection of low-abundance viruses, false negatives / false positives, and fail to meet the regulatory compliance requirements for pharmaceuticals. This invention employs a standardized detection process (S1-S5). First, samples undergo freeze-thaw lysis and centrifugation. Repeated freeze-thaw cycles disrupt cell structure to fully release intracellular viruses, while centrifugation removes cell debris and other impurities, preparing a sample processing solution free of interference. This provides a pure matrix foundation for subsequent cell culture and virus detection. Next, based on the sample source and detection target, corresponding susceptible indicator cell lines are precisely matched. Cells are plated and cultured to 30-60% confluence to ensure cell viability and viral infection efficiency. Four control groups (NC, EC, PC, IG) are set up to eliminate non-specific interference from the sample matrix and verify the method's specificity and anti-interference ability. After incubation, the medium is replaced with complete culture medium and cultured continuously for at least 21 or 28 days, passaged at preset intervals. Utilizing the mechanism of long-term culture and blind passage, efficient proliferation and enrichment of trace amounts and low abundance viruses in the sample are achieved. CPE is observed throughout the process. It can capture cell morphological changes caused by viral infection in real time; after the culture cycle, it uses a combination of endpoints such as HAD, HA, immunofluorescence staining, and quantitative detection of reverse transcriptase activity to verify the mechanism from multiple dimensions such as cytopathic effects, viral hemagglutination characteristics, specific antigen recognition, and viral enzyme activity, avoiding the limitations of single detection methods and comprehensively improving the sensitivity and accuracy of virus detection; finally, it performs positive and negative determination based on the results of multi-endpoint detection and completes the methodology validation. Relying on the mechanism of complete methodology validation, it ensures that the specificity, detection limit, precision, and robustness of the detection method meet the regulatory requirements of ChP, USP, ICH, etc., and ultimately achieves highly reliable, high-coverage, and compliant detection of viral factors in biological products.

[0006] The methodology validation parameters include at least specificity, detection limit, repeatability, intermediate precision, and robustness.

[0007] In step S1, the freeze-thaw lysis involves freezing the sample at -80℃±10℃ and thawing it completely in a water bath at 37℃±1℃, repeating this cycle 3 times; the centrifugation process involves centrifuging at 4℃ and 2000×g for 15 minutes and collecting the supernatant.

[0008] In step S2, when the detection target is a broad-spectrum exogenous viral factor in a biological product, the indicator cell line is selected from at least three of Vero cells, MRC-5 cells, CHO-K1 cells, HEK-293 cells, and HeLa cells; the indicator virus used in the positive control group and the interference group is selected from at least one of human parainfluenza virus type 3 (PI-3), encephalomyocarditis virus (EMC), vesicular stomatitis virus (VSV), and human adenovirus type 5 (Adeno-5).

[0009] In step S3, the subculture operation of the preset node specifically includes: subculturing on the 7th, 14th and 21st days of the culture period; In step S3, the passage operation of the preset node specifically includes: passage on day 7 and day 21 of culture, and seeding the cell culture supernatant and / or cell lysis supernatant into new indicator cells for secondary expansion culture on day 14.

[0010] This study found that a two-stage inoculation and amplification strategy of 14 days of primary culture followed by 14 days of secondary culture, with simultaneous collection of cell culture supernatant and cell lysis supernatant on day 14 of primary culture, was used to inoculate new indicator cell lines for secondary amplification culture. This two-stage continuous culture amplification achieved efficient enrichment of trace amounts of virus in the sample. Combined with two blind passage operations on days 7 and 21 of the culture cycle, the viral proliferation cycle was further extended. With the addition of multi-dimensional endpoint detection methods, the detection capability of low-abundance and low-infectivity viral factors was significantly improved. The method validation results showed that the lowest detection concentration of various characteristic viruses under this strategy was superior to that of traditional single-culture methods, providing a more reliable technical guarantee for the viral safety detection of high-risk samples such as terminal cells in biopharmaceutical production and master / working cell banks.

[0011] In step S4, the multi-endpoint joint detection is a combination of the hemoadsorption assay (HAd) and the erythrocyte agglutination assay (HA). The hemoadsorption assay uses a 0.2% to 0.5% concentration of a mixture of guinea pig, chicken, and human erythrocytes, which is incubated at 2 to 8°C and 20 to 25°C for 30 minutes, respectively, and then examined under a microscope. The erythrocyte agglutination assay uses a 0.2% to 0.5% concentration of a mixture of guinea pig, chicken, and human erythrocytes, which is first incubated at 2 to 8°C for 30 minutes, and then transferred to 20 to 25°C for 30 minutes. The agglutination results are then observed.

[0012] In step S2, when the detection target is a bovine-specific virus, the indicator cell line is selected from at least one of MDBK cells, Vero cells, MRC-5 cells, and BT cells; the indicator viruses used in the positive control group and the interference group are selected from at least one of bovine viral diarrhea virus (BVDV), reovirus type 3 (Reo-3), bovine parainfluenza virus type 3 (BPIV-3), rabies virus (RV), bovine respiratory syncytial virus (BRSV), bovine adenovirus (BAV), and bluetongue virus (BTV); in step S4, the multi-endpoint joint detection is a combination of CPE observation and immunofluorescence staining detection.

[0013] In step S2, when the detection target is a porcine-specific virus, the indicator cell line is selected from at least one of BT cells, MRC-5 cells, and ST cells; the indicator virus used in the positive control group and the interference group is selected from at least one of Reovirus 3 (Reo-3), porcine parvovirus (PPV), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), rabies virus (RV), porcine transmissible gastroenteritis virus (TGEV), and porcine adenovirus (PAV); in step S4, the multi-endpoint joint detection is a combination of CPE observation and immunofluorescence staining detection.

[0014] In step S2, when the target of detection is an infectious retrovirus, the indicator cell line is SC-1 mouse embryonic cells; the indicator virus used in the positive control group and the interference group is tropism-dependent mouse leukemia virus (E-MuLV); in step S3, the cells are passaged every 3-4 days during continuous culture, for a total of 5 passages and a cumulative culture period of 21 days; in step S4, the multi-endpoint joint detection is a quantitative detection of reverse transcriptase activity based on Q-PERT technology.

[0015] In step S5, the acceptable criteria for the methodology validation parameters are specifically as follows: Specificity: The positive control group and interference group tested positive, while the negative control group and experimental group showed no detection of the target virus. Limit of detection: The lowest limit of detection for the target virus is not higher than 10 TCID. 50 (Half-maximal dose for tissue cell infection) or 10 copies. Repeatability: The results of six repeated tests on the same sample by the same researcher were consistent; Intermediate precision: The results of six tests performed on the same sample by two researchers at different times were consistent; Durability: The test results are unaffected by minor, preset changes in the test parameters.

[0016] The biological product samples to be tested include any one of the following: cell bank samples, unpurified cell harvest fluid, terminal production cells, bovine / porcine biological raw materials, vaccines, and recombinant protein biological products.

[0017] Beneficial effects 1. The method of this invention can avoid the limitations of a single detection method and comprehensively improve the sensitivity and accuracy of virus detection. At the same time, it can determine the positive and negative results based on the multi-endpoint detection results and complete the methodological validation. Relying on the mechanism of complete methodological validation, it ensures that the specificity, detection limit, precision and robustness of the detection method meet the regulatory requirements of ChP, USP, ICH and other regulations, and finally achieve highly reliable, high-coverage and compliant detection of viral factors in biological products.

[0018] 2. Based on the matrix characteristics and potential contaminating virus types of different biological products, this method establishes a precise indicator cell selection system, matching multiple sets of indicator cell combinations for recombinant protein and antibody biological products, covering susceptible cells from various genera of viruses such as human, monkey, and mouse.

[0019] 3. This invention achieves high coverage detection of a broad spectrum of endogenous and exogenous viral factors in biological products by using two blind passages within a 28-day culture cycle and inoculating the supernatant and cell lysis supernatant on day 14, combined with CPE observation for multi-dimensional detection, thus solving the industry pain point of limited detection range of single cell lines.

[0020] 4. The detection method of this invention can reduce the detection limit of bovine viruses to 1 TCID. 50 / ml, and the specificity of the method was verified by matrix interference test, which greatly reduced the risk of missed detection of low titer virus contamination. The test results fully comply with the virus safety control requirements of the Chinese Pharmacopoeia and the United States Pharmacopeia for bovine biological products.

[0021] 5. The detection method of this invention can achieve a detection limit of 1 TCID for all five porcine characteristic viruses. 50 / ml, it maintains stable results even under varying conditions such as different red blood cell ratios and antibody incubation temperatures, effectively resisting non-specific interference from the sample matrix, and can be stably applied to the virus safety detection of various samples such as porcine cell banks, veterinary vaccines, and porcine raw materials. Detailed Implementation

[0022] Example 1 A method for detecting viruses in biological products based on indicator cell culture includes the following steps: (1) Sample pretreatment: CHO-K1 cell suspension was frozen at -80℃±10℃ and thawed in a water bath at 37℃±1℃. The freeze-thaw cycle was repeated 3 times. The sample was centrifuged at 4℃ and 2000×g for 15 minutes, and the supernatant was collected to obtain the sample lysis supernatant.

[0023] (2) Virus dilution: The original PI-3, EMC, and VSV virus solutions were serially diluted with MEM medium / sample lysis supernatant to prepare 1 TCID. 50 / ml, 10TCID 50 / ml, 100TCID 50 Three concentration gradients per milliliter.

[0024] (3) Construction of the seeding system: Vero, MRC-5, and CHO-K1 cells were seeded in 6-well plates and cultured to a confluence of 30%-60%. The following groups were set up, with 1 ml of treatment solution seeded in each well and incubated at 37℃ and 5% CO2 for 120±10 minutes: NC group: Vero, MRC-5, and CHO-K1 cells were seeded in 1 ml of MEM, MEM, and Ham's F-12K culture media per well, respectively. Each cell type was seeded in one 6-well plate, with a total of 6 wells seeded; EC group: Vero, MRC-5, and CHO-K1 cells were seeded in 1 ml of sample lysis supernatant per well, with a total of 6 wells seeded for each cell type. One 6-well plate was used, with a total of 6 wells seeded. For the PC group: Vero cells were seeded with 1 ml of PI-3 virus per well; MRC-5 cells were seeded with 1 ml of EMC virus per well; and CHO-K1 cells were seeded with 1 ml of VSV virus per well. Each cell type and corresponding virus was seeded onto one 6-well plate. For the IG group: Vero cells were seeded with 1 ml of sample lysis supernatant containing PI-3 virus per well; MRC-5 cells were seeded with 1 ml of sample lysis supernatant containing EMC virus per well; and CHO-K1 cells were seeded with 1 ml of sample lysis supernatant containing VSV virus per well. Each cell type and corresponding virus was seeded onto one 6-well plate.

[0025] After each group of indicator cells was seeded, it was incubated in a 37℃±1℃, 5%±1%CO2 incubator for 120±10 minutes.

[0026] (4) Amplification culture: After incubation, discard the inoculum and add 2 ml of complete culture medium containing FBS to each well (Vero and MRC-5 use 5% FBS+MEM, and CHO-K1 uses 2% FBS+Ham's F-12K). Culture continuously at 37℃ and 5% CO2 for 28 days. During the culture period, passage is performed on the 7th, 14th and 21st days respectively, and CPE is observed under a microscope at least twice every 7 days.

[0027] (5) Endpoint detection: On day 28 of culture, HAD was performed on cells in each group, and the culture supernatant was used for HA. HAD and HA were both prepared using 0.2% and 0.5% guinea pig + chicken + human red blood cell mixture and guinea pig + chicken red blood cell mixture, respectively. HAD was incubated at 2~8℃ and 20~25℃ for 30 minutes and the results were observed. HA was first incubated at 2~8℃ for 30 minutes and then at 20~25℃ for 30 minutes and the results were observed.

[0028] (6) Method validation: Perform specificity, detection limit, repeatability, intermediate precision, and robustness validation according to the above protocol. Throughout the process, observe for CPE, and at the end of the observation period, collect cell cultures for hemoadsorption and collect cell culture supernatant for HAD. Determine whether the test sample contains viral factors by observing whether indicator cells produce CPE, HAD, and red blood cell agglutination.

[0029] Equipment: HR40-IIA2 biosafety cabinet, Herocell180CO2 incubator, XD-202 microscope, 5810R centrifuge, -80℃ ultra-low temperature freezer; Indicator cells: African green monkey kidney cells (Vero, ATCC CCL-81), human embryonic lung fibroblasts (MRC-5, ATCCCCL-171), and mouse ovarian epithelial cells (CHO-K1, ATCC CCL-61). Indicator viruses: Human parainfluenza virus type 3 (PI-3, ATCC VR-93), encephalomyocarditis virus (EMC, ATCC VR-129B), vesicular stomatitis virus (VSV, ATCC VR-3340). Reagents: MEM medium, Hams F-12K medium, fetal bovine serum (FBS), 0.25% trypsin EDTA, anticoagulated guinea pig blood, anticoagulated chicken blood, and human anticoagulated whole blood; Sample to be tested: CHO-K1 cell suspension (cell density ≥1×10⁻⁶) 7 (each / ml).

[0030] Example 2 The specific implementation method is the same as in Example 1; the difference is that the indicator cells are: African green monkey kidney cells (Vero), human embryonic lung fibroblasts (MRC-5), human cervical cancer cells (HeLa), and human embryonic kidney cells (HEK-293); the indicator viruses are: human parainfluenza virus type 3 (PI-3), encephalomyocarditis virus (EMC), and human adenovirus type 5 (Adeno-5); and the test sample is: HEK-293 cell suspension (cell density ≥1×10⁻⁶). 7 (each / ml).

[0031] NC group: 1 ml of MEM, MEM, MEM and DMEM culture medium was seeded into each well of the four cell types Vero, MRC-5, HeLa and HEK-293 respectively, with each cell type in a 6-well plate.

[0032] EC group: 1 ml of sample lysis supernatant was seeded into each well of the four cell types Vero, MRC-5, HeLa and HEK-293, with each cell type in a 6-well plate.

[0033] PC group: Vero cells were inoculated with 1 ml of PI-3 virus per well; MRC-5 cells were inoculated with 1 ml of EMC virus per well; HeLa cells were inoculated with 1 ml of EMC virus per well; HEK-293 cells were inoculated with 1 ml of Adeno-5 virus per well; a total of one 6-well plate was inoculated with each cell type and corresponding virus.

[0034] IG group: Vero cells were seeded in each well with 1 ml of sample lysis supernatant containing PI-3 virus; MRC-5 cells were seeded in each well with 1 ml of sample lysis supernatant containing EMC virus; HeLa cells were seeded in each well with 1 ml of sample lysis supernatant containing EMC virus; HEK-293 cells were seeded in each well with 1 ml of sample lysis supernatant containing Adeno-5 virus; a total of one 6-well plate was seeded for each cell type and corresponding virus.

[0035] After each group of indicator cells was seeded, it was incubated in a 37℃±1℃, 5%±1%CO2 incubator for 120±10 minutes.

[0036] Example 3 The specific implementation method is the same as in Example 1; the difference is that the amplification culture is as follows: continuous culture at 37℃ and 5% CO2 for 28 days, and passage is performed on the 7th and 21st days during the culture period; on the 14th day, the cell culture supernatant and cell lysis supernatant of each group are collected and seeded into new corresponding indicator cells for secondary amplification culture. The secondary culture system is consistent with the original system; CPE is observed under a microscope at least twice every 7 days throughout the process.

[0037] Example 4 The specific implementation method is the same as in Example 2; the difference is that the amplification culture is as follows: continuous culture at 37℃ and 5% CO2 for 28 days, and passage is performed on the 7th and 21st days during the culture period; on the 14th day, the cell culture supernatant and cell lysis supernatant of each group are collected and seeded into new corresponding indicator cells for secondary amplification culture. The secondary culture system is consistent with the original system; CPE is observed under a microscope at least twice every 7 days throughout the process.

[0038] Example 5 A method for detecting viruses in biological products based on indicator cell culture includes the following steps: (1) Sample pretreatment: After the sample to be tested was lysed by three freeze-thaw cycles, it was centrifuged at 4℃ and 2000×g for 15 minutes to collect the supernatant and diluted with the corresponding culture medium for later use; (2) Construction of the inoculation system: Vero, MRC-5, MDBK and BT cells were seeded in T75 cell culture flasks and cultured until the confluence was 30-60%. NC group, EC group, PC group and IG group were set up. For each type of indicator cell, one T75 flask was taken, the culture medium in the flask was discarded, and 3 ml of the corresponding basal culture medium was inoculated and recorded as NC group; another T75 flask was taken, the culture supernatant was discarded, and 3 ml of sample (cell lysis buffer) was inoculated and recorded as EC group; the NC group and EC group culture flasks were placed in a 37℃±1℃, 5%±1%CO2 incubator for 120 minutes±10 minutes, and then 9 ml of complete culture medium containing 5%FBS was added and cultured for 7 days. During the culture period, the medium was changed or replenished according to the cell status.

[0039] (3) Amplification culture: After the incubation, replace the medium with MEM containing 2% FBS and culture continuously for 14 days. On the 7th day of the period, passage is performed. On the 14th day of culture, the PC group is inoculated with positive control virus according to Table 1 below. 500 μL is inoculated per well and placed in a 37℃±1℃, 5%±1% CO2 incubator for 120 minutes±10 minutes. Then, add complete medium containing 5% FBS to about 3 ml. Observe CPE under a microscope every day.

[0040] Table 1

[0041] *Note: A positive slide is used when performing fluorescent antibody testing for rabies virus (RV).

[0042] (4) Endpoint detection: Cultured for 21 days, during which medium was changed or replenished according to the cell state. HAD was tested, and specific fluorescence was observed under a fluorescence microscope. On day 21 of the experiment: fluorescent antibody detection was performed. Indicator cells of each group were taken, and two wells were selected for each virus to be detected. After labeling, the supernatant was discarded, and 500 μL of 14% paraformaldehyde was added for fixation for 15 minutes. The cells were washed 3 times with PBS. 500 μL of 0.5% Triton-100 was added, and the cells were permeated at room temperature for 15 minutes. The cells were washed 3 times with PBS. 500 μL of 1% BSA blocking solution was added, and the cells were blocked at room temperature for 30 minutes. 500 μL of the corresponding fluorescent antibody or diluent was added in a dark environment, and the cells were incubated at room temperature for 60 minutes or at least 12 hours. The cells were washed 3 times with PBS, and the fluorescence was observed under a fluorescence microscope. If pre-prepared slides were used, no pretreatment was required. 100 μL of the corresponding fluorescent antibody or diluent was added to the slide, and the cells were incubated at room temperature for 60 minutes or at least 12 hours. The cells were washed 3 times with PBS, and the fluorescence was observed under a fluorescence microscope.

[0043] (5) Method validation: Complete the validation of specificity, detection limit, repeatability, intermediate precision and robustness.

[0044] Equipment: Same as in Example 1, except for the addition of a fluorescence microscope; Indicator cells: Vero cells, MRC5 cells, MDBK cells, BT cells; Indicator viruses: Reo-3 virus, BVDV virus, BPIV-3 virus, BPV virus, BAV virus, BTV virus, BRSV virus; Reagents: MEM culture medium, fetal bovine serum (FBS), BVDV / RV / Reo-3 / BRSV / BAV / BTV / BPV / BPIV-3 specific fluorescent antibodies, DAPI staining solution; Sample to be tested: Bovine cell-derived sample (cell density ≥ 1 × 10⁻⁶) 7 (each / ml).

[0045] Example 6 A method for detecting viruses in biological products based on indicator cell culture includes the following steps: (1) Sample pretreatment: After the sample to be tested was lysed by three freeze-thaw cycles, the supernatant was collected by centrifugation at 4℃ and 2000×g for 15 minutes and diluted with the corresponding culture medium for later use; (2) Construction of inoculation system: Vero, BT, and ST were cultured in T75 culture flasks to ensure that the cell confluence was 30%-60% on the day of the experiment. NC group, EC group, PC group, and IG group were set up. After inoculation with the corresponding treatment solution, the cells were incubated at 37℃ and 5%CO2 for 120 minutes ± 10 minutes; (3) Amplification culture: After the incubation, the maintenance medium was replaced and cultured continuously for 14 days. The cells were passaged on the 7th day. The PC group was inoculated with the positive control virus according to Table 2 below. 500 μL of virus was inoculated in each well and incubated in a 37℃±1℃ and 5%±1%CO2 incubator for 120 minutes ± 10 minutes. Then, complete culture medium containing 5%FBS was added to about 3 ml and cultured for 21 days. During this period, the medium was changed or replenished according to the cell status. The CPE was observed by microscopic examination every day. Table 2

[0046] *Note: A positive slide is used when performing fluorescent antibody testing for rabies virus (RV).

[0047] (4) Endpoint detection: cultured for 21 days, during which the medium was changed or replenished according to the cell status, and HAD was observed under a fluorescence microscope to detect specific fluorescence (same as in Example 5); (5) Methodological verification (same as in Example 5).

[0048] Equipment: Same as in Example 5; Indicator cells: Vero cells, BT cells, ST cells; Indicator viruses: PPV virus, PAV virus, TGEV virus, BVDV virus, Reo-3 virus; Reagents: MEM medium, DMEM medium, fetal bovine serum (FBS), PPV / PAV / TGEV / BVDV / Reo-3 / RV specific fluorescent antibodies; Sample to be tested: Porcine-derived cell samples (cell density ≥ 1 × 10⁻⁶) 7 (each / ml).

[0049] Example 7 A method for detecting viruses in biological products based on indicator cell culture includes the following steps: (1) Sample pretreatment: Prepare sample lysis supernatant as in Example 1; (2) Virus dilution: Serially dilute the original E-MuLV virus solution with EMEM medium / sample lysis supernatant to prepare 10 copies, 10 2 copies, 10 3 Three concentration gradients of copies; (3) Construction of seeding system: SC-1 cells were plated to a confluence of 30%-60%, and NC group, EC group, PC group and IG group were set up. 1 ml of the corresponding treatment solution containing 8 μg / ml Polybrene was added to each well, and the cells were incubated at 37℃ and 5% CO2 for 120±10 minutes; (4) Amplification culture: After incubation, the EMEM complete medium containing 5% FBS was replaced and cultured continuously for 21 days. The cells were passaged once every 3-4 days for a total of 5 passages; (5) Endpoint detection: 3-4 days after the 5th passage, the cell culture supernatant of each group was collected, and the reverse transcriptase activity was quantitatively detected by Q-PERT (reverse transcriptase activity test method). The Ct value was obtained by real-time fluorescence quantitative PCR to determine the reverse transcriptase activity; (6) Method validation: Validation of specificity, detection limit, repeatability, intermediate precision and robustness were completed.

[0050] Equipment: Same as in Example 1, except for the addition of an ABI 7500 real-time quantitative PCR instrument; Indicator cells: SC-1 mouse embryonic cells (ATCC); Indicator virus: tropism-positive mouse leukemia virus (E-MuLV, ATCC); Reagents: EMEM medium, Polybrene, fetal bovine serum (FBS), Q-PERT detection primers and probes, MS2 RNA, ProbeqPCRMix; Sample to be tested: CHO-K1 cell suspension (cell density ≥1×10⁻⁶) 7 (each / ml).

[0051] Performance testing methods and data 1. The methodological validation results of Example 1 are shown in Table 3: (1) Specificity verification: No CPE was observed in the NC and EC groups throughout the entire process, and the HAD and HA results were negative; CPE was observed in all wells of the PC and IG groups after inoculation with 100 TCID50 / well, and the HAD and HA results were positive, thus the specificity verification was passed. (2) Detection limit verification: Stable CPE was observed in both the PC and IG groups for PI-3, EMC, and VSV at an inoculation dose of 1 TCID50 / well. Among them, the HAD and HA results of Vero cells infected with PI-3 were positive, and the method's lowest detection limit for the three viruses reached 1 TCID50. 50 (3) Repeatability and intermediate precision: The results of 6 repeated tests by the same experimenter and 6 tests by two experimenters at different times were completely consistent. The NC / EC group was negative and the PC / IG group was positive. The repeatability and intermediate precision were verified. (4) Robustness: When the red blood cell types and concentrations varied in the range of 0.2%-0.5%, the HAD and HA detection results were not different. The robustness was verified.

[0052] Table 3

[0053] 2. The methodological verification results of Example 2 are shown in Table 4: (1) Specificity verification: No CPE was observed in the NC and EC groups throughout the entire process, and the HAD and HA results were negative; CPE was observed in all PC and IG groups after inoculation with 100 TCID50 / well, and the HAD and HA results were positive, thus the specificity verification was passed. (2) Detection limit verification: Stable CPE was observed in both the PC and IG groups for PI-3, EMC, and Adeno-5 viruses at an inoculation dose of 1 TCID50 / well. Among them, the HAD and HA results of Vero cells infected with PI-3 were positive, and the method's lowest detection limit for the three viruses reached 1 TCID50. 50 Better than the regulatory requirement of ≤10 TCID 50 Standard. (3) Repeatability and intermediate precision: The results of 6 repeated tests by the same experimenter and 6 tests by two experimenters at different times were completely consistent. The NC / EC group was negative and the PC / IG group was positive. The repeatability and intermediate precision were verified. (4) Robustness: When the red blood cell types and concentrations varied in the range of 0.2%-0.5%, the HAD and HA detection results were not different. The robustness was verified.

[0054] Table 4

[0055] 3. The methodological verification results of Example 3 are shown in Table 5: (1) Specificity verification: No CPE was observed in the primary culture and secondary inoculation culture of the NC group and EC group, and the results of HAD and HA were negative; 100 TCID in the PC group and IG group 50 / After inoculation with the pore virus, stable CPE was observed in both primary and secondary cultures, and the results for HAD and HA were both positive, thus the specificity verification was successful. (2) Detection limit verification: PI-3, EMC, and VSV viruses were detected at 1 TCID. 50 At the inoculum size of / well, the CPE detection rate after secondary inoculation was 100%, and typical cytopathic effects were observed 2-3 days earlier than the conventional protocol. The sensitivity for detecting low-titer viruses was further improved, while the limit of detection still reached 1 TCID. 50 (3) Repeatability, intermediate precision and durability all meet acceptable standards and have been verified.

[0056] Table 5

[0057] 4. The methodological verification results of Example 4 are shown in Table 6: (1) Specificity verification: No CPE was observed in the primary culture and secondary inoculation culture of the NC group and EC group, and the results of HAD and HA were negative; 100 TCID in the PC group and IG group 50 / After inoculation with the pore virus, stable CPE was observed in both primary and secondary cultures, and the results for HAD and HA were both positive, thus the specificity verification was successful. (2) Detection limit verification: PI-3, EMC, and Adeno-5 viruses were detected at 1 TCID. 50 At the inoculum size of / well, the CPE detection rate after secondary inoculation was 100%, and typical cytopathic effects were observed 2-3 days earlier than the conventional protocol. The sensitivity for detecting low-titer viruses was further improved, while the limit of detection still reached 1 TCID. 50 (3) Repeatability, intermediate precision and durability all meet acceptable standards and have been verified.

[0058] Table 6

[0059] 5. The methodological validation results of Example 5 are shown in Table 7: (1) Specificity verification: No CPE was observed in the NC and EC groups, and no specific fluorescence was observed during fluorescent staining; typical CPE of the corresponding virus was observed in the PC and IG groups, and specific bright green fluorescence was observed during fluorescent staining, thus the specificity verification was passed. (2) Detection limit verification: The method achieved a minimum detection limit of 1 TCID for BVDV, Reo-3, BPV, BAV, BPIV-3, BTV, and BRSV. 50(3) Repeatability and intermediate precision: The results of 6 repeated tests and the results of the two testers were completely consistent, with no false positives / false negatives, and the verification was passed. (4) Robustness: When the cell seeding confluence varied in the range of 30%-80% and the antibody incubation time varied in the range of 30-60 minutes, the test results were not different, and the robustness verification was passed.

[0060] Table 7

[0061] 6. The methodological verification results of Example 6 are shown in Table 8: (1) Specificity verification: No CPE or specific fluorescence was observed in the NC and EC groups; typical CPE of the corresponding viruses was observed in the PC and IG groups, and specific positive signals were visible in the fluorescent staining, thus the specificity verification was passed. (2) Detection limit verification: The method's lowest detection limit for BVDV, Reo-3, PPV, PAV, and TGEV can all reach 1 TCID50, meeting the detection requirements. (3) Repeatability, intermediate precision, and robustness all meet the preset acceptable standards, and all verifications were passed.

[0062] Table 8

[0063] 7. The methodological validation results of Example 7 are shown in Table 9: (1) Specificity verification: No reverse transcriptase activity was detected in the NC group and EC group; 10 in the PC group and IG group 2 High levels of reverse transcriptase activity (2.05 × 10⁻⁶) were detected in all copies of E-MuLV after inoculation. 11 pU / mL ~3.73×10 11 (1) pU / mL), specificity verification passed. (2) Detection limit verification: After 10 copies of E-MuLV were inoculated, reverse transcriptase activity could be stably detected in both the PC group and the IG group. The lowest detection limit of the method can reach 10 copies, which meets the detection requirements. (3) Repeatability and intermediate precision: The results of 6 repeated tests by the same experimenter and the results of tests by two experimenters at different times were consistent, with no false negatives / false positives, and the verification passed. (4) Robustness: When the SC-1 cell passages changed in the P8-P11 range, the detection results were not significantly different, and the robustness verification passed.

[0064] Table 9

Claims

1. A method for detecting viruses in biological products based on indicator cell culture, characterized in that, Includes the following steps: S1, freeze-thaw lysis and centrifugation are performed on the biological product sample to be tested to prepare the sample processing solution; S2. Based on the source of the sample and the detection target, the corresponding indicator cell line is matched. After the indicator cells are plated and cultured to a confluence of 30-60%, NC group, EC group, PC group and IG group are set up, and the corresponding treatment solutions are inoculated and incubated respectively. S3, after incubation, replace with complete culture medium and carry out continuous culture for 21 or 28 days. During the culture period, perform subculture according to the preset nodes and observe CPE throughout the process. S4. After the culture cycle is completed, endpoint tests are performed on each group of cells. The endpoint tests include HAd, HA, immunofluorescence staining detection, and quantitative detection of reverse transcriptase activity. S5. Based on the results of multi-endpoint detection, determine the positive or negative status of the sample and conduct methodological validation.

2. The detection method according to claim 1, characterized in that, The method validation methods include specificity, detection limit, repeatability, intermediate precision, and robustness.

3. The detection method according to claim 1, characterized in that, The detection targets are broad-spectrum exogenous viral factors, bovine-derived specific viruses, porcine-derived specific viruses, and infectious retroviruses in biological products.

4. The detection method according to claim 1, characterized in that, In step S2, the detection target is a broad-spectrum exogenous viral factor in the biological product, and the indicator cell line is selected from at least three of Vero cells, MRC-5 cells, CHO-K1 cells, HEK-293 cells, and HeLa cells; the indicator virus used in the positive control group and the interference group is selected from at least one of PI-3, EMC, VSV, and Adeno-5.

5. The detection method according to claim 1, characterized in that, In step S3, the subculture operation of the preset node specifically includes: subculturing on the 7th, 14th and 21st days of the culture period.

6. The detection method according to claim 1, characterized in that, In step S3, the passage operation of the preset node specifically includes: passage on day 7 and day 21 of culture, and seeding the cell culture supernatant or cell lysis supernatant into new indicator cells for secondary expansion culture on day 14.

7. The detection method according to claim 6, characterized in that, In step S2, when the detection target is a bovine-specific virus, the indicator cell line is selected from at least one of MDBK cells, Vero cells, MRC-5, and BT cells; the indicator viruses used in the positive control group and the interference group are selected from at least one of bovine viral diarrhea virus, reovirus type 3, bovine respiratory syncytial virus, bovine parainfluenza virus type 3, rabies virus, bovine adenovirus, bluetongue virus, and bovine parvovirus.

8. The detection method according to claim 1, characterized in that, In step S2, when the detection target is a porcine-derived specific virus, the indicator cell line is selected from at least one of BT cells, Vero cells, and ST cells; the indicator virus used in the positive control group and the interference group is selected from at least one of reovirus type 3, porcine parvovirus, bovine viral diarrhea virus, bovine parainfluenza virus type 3, rabies virus, porcine transmissible gastroenteritis virus, and porcine adenovirus.

9. The detection method according to claim 1, characterized in that, In step S2, when the detection target is an infectious tropism retrovirus, the indicator cell line is SC-1 mouse embryonic cells; the indicator virus used in the positive control group and the interference group is tropism mouse leukemia virus.

10. The detection method according to claim 1, characterized in that, The biological product samples to be tested include one of the following: cell bank samples, unpurified cell harvest fluid, terminal production cells, bovine biological raw materials, porcine biological raw material vaccines, and recombinant protein biological products.

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